Cloning, characterization and specificity of a new aromatic-L-amino-acid decarboxylases from Bufo bufo gargarizans.

5-Hydroxytryptamine (5-HT) and its derivative bufotenine, which possess important physiological functions, are the primary active components in the secretions of toad parotid and skin gland. However, the biosynthetic pathway of these substances remains unclear in toads. To characterize toad's Aromatic-L-amino-acid decarboxylase (AADC), the key enzyme in the predicted 5-HT derivatives biosynthetic pathway, the full-length cDNA of AADC from Bufo bufo gargarizans (BbgAADC) was cloned from the parotoid gland of B. bufo gargarizans. The recombinant BbgAADC exhibited optimal expression in E. coli BL21 (DE3) containing pCold-BbgAADC after induction for 16 h at 15 °C with 0.3 mM IPTG, resulting in substantial yields of soluble proteins. The enzymological properties of BbgAADC were assessed, and it was determined that the optimal reaction temperature was 37 °C, the optimal pH was 8.6, and the optimum molar ratio of pyridoxal-5'-phosphate (PLP) to BbgAADC was found to be 3.6:1. Additionally, high substrate specificity was observed, as BbgAADC could catalyze the production of 5-HT from 5-hydroxytryptophan (5-HTP) but not dopamine or tryptamine from levodopa or tryptophan, respectively. The Km of the recombinant protein BbgAADC was 0.2918 mM and the maximum reaction rate (Vmax) was 1.182 µM·min-1 when 5-HTP was used as substrate. The Kcat was 0.0545 min-1, and Kcat/Km was 0.1868 mM-1·min-1. To elucidate the mechanism of BbgAADC, molecular docking was performed with PLP and 5-HTP, or the external aldimine formed by 5-HTP and PLP. The results indicated that the active sites for BbgAADC to bind with PLP were K303, H192, N300, A148, F309, T246, A273, and T147. W71, Y79, F80, P81, T82, H192, T246, N300, H302, F309, and R477 served as catalytically active sites for the binding of BbgAADC to 5-HTP. Furthermore, R447, W71, S149, N300, A148, and T147 of BbgAADC were involved in the decarboxylation reaction of the aldimine formed by PLP and 5-HTP.

Tunable Toxicity of Bufadienolides is Regulated through a Configuration Inversion Catalyzed by a Short-Chain Dehydrogenase/Reductase.

Bufadienolides are toxic components widely found in amphibious toads that exhibit a wide range of biological activities. Guided by UPLC-QTOF-MS analysis, several 3-epi-bufadienolides with unique structures were isolated from the bile of the Asiatic toad, Bufo gargarizans. However, the enzymatic machinery of this epimerization in toads and its significance in chemical ecology remains poorly understood. Herein, we firstly compared the toxicities of two typical bufadienolides, bufalin (featuring a 14ß-hydroxyl) and resibufogenin (containing a 14, 15-epoxy group), with their corresponding 3-epi isomers in a zebrafish model. The results of the toxicology assays showed that the ratio of maximum non-toxic concentrations of these two pairs of compounds are 256 and 96 times, respectively, thereby indicating that 3-hydroxyl epimerization leads to a significant decrease in toxicity. Aiming to investigate the biotransformation of 3-epi bufadienolides in toads, we applied liver lysate to transform bufalin and found that it could stereoselectively catalyze the conversion of bufalin into its 3α-hydroxyl epimer. Following this, we cloned and characterized a short-chain dehydrogenase/reductase, HSE-1, from the toad liver cDNA library and verified its 3(ß→α)-hydroxysteroid epimerization activity. To the best of our knowledge, this is the first hydroxyl epimerase identified from amphibians that regulates the toxicity of animal-derived natural products.

Cloning and characterization of steroid 5ß-reductase from the venom gland of Bufo bufo gargarizans.

Bufadienolides are the main active ingredients of Venenum Bufonis, which is a widely used traditional Chinese medicine secreted from parotoid gland and skin glands of Bufo bufo gargarizans. According to the transcriptome analysis, "cholesterol-bile acid-bufadienolidies pathway" was proposed as animal-derived bufadienolides biosynthesis pathway by us previously. In this pathway 3ß-hydroxysteroid dehydrogenase (3ßHSD) and steroid 5ß-reductase (SRD5ß) might be the key enzymes to convert the A/B ring to cis-configuration. Therefore, as the second report of our group, here we report the cloning of the full length of SRD5ß cDNA of B. bufo gargarizans (Bbg-SRD5ß) from the parotoid gland of B. bufo gargarizans for the first time, and site-directed mutagenesis was used to explored the character of Bbg-SRD5ß. Bbg-SRD5ß had an open reading frame of 981 bp and encoded 326 amino acids residues. The expression conditions of the recombinant Bbg-SRD5ß in E. coli BL21 (DE3) harbored with pCold-Bbg-SRD5ß was optimized as induction for 10 h at 15 °C with 0.1 mM IPTG. With NADPH as a cofactor, Bbg-SRD5ß can reduce the Δ4,5 double bonds of progesterone to generate dihydroprogesterone õwithout substrate inhibition effect. The catalytic rate of mutant type Bbg-SRD5ß-Y132G was 1.8 times higher than that of wild type Bbg-SRD5ß. Although Bbg-SRD5ß was almost unable to reduce the progesterone to dihydroprogesterone after mutation of V309, the affinity of enzyme with NADPH changed significantly. Bbg-SRD5ß is the key enzymes to convert the A/B ring of steroid to cis-configuration, and V309 is a key site affecting the binding affinity of enzyme with NADPH, and the mutation of Y132 can adjust the catalytic rate of Bbg-SRD5ß.

Two New Indole Alkaloids from Toad Venom of Bufo bufo gargarizans.

Two new indole alkaloids, Bufotenidine B (2) and Bufocarboline A (6), along with seven known indole alkaloids (1, 3-5, and 7-9) and three organic acids (10-12), were isolated from the water extract of toad venom. The structures of the new alkaloids were elucidated by extensive spectroscopic methods. The absolute configurations of 4, 6, and 8 were determined for the first time by electronic circular dichroism (ECD) calculations. The cytotoxic activity of all compounds was tested against human malignant melanoma cells A375 by the MTT method, and no antitumor activity was observed.

The impact of multiple climatic and geographic factors on the chemical defences of Asian toads (Bufo gargarizans Cantor).

Chemical defences are widespread in nature, yet we know little about whether and how climatic and geographic factors affect their evolution. In this study, we investigated the natural variation in the concentration and composition of the main bufogenin toxin in adult Asian toads (Bufo gargarizans Cantor) captured in twenty-two regions. Moreover, we explored the relative importance of eight climatic factors (average temperature, maximum temperature, minimum temperature, average relative humidity, 20-20 time precipitation, maximum continuous precipitation, maximum ground temperature, and minimum ground temperature) in regulating toxin production. We found that compared to toads captured from central and southwestern China, toads from eastern China secreted higher concentrations of cinobufagin (CBG) and resibufogenin (RBG) but lower concentrations of telocinobufagin (TBG) and cinobufotalin (CFL). All 8 climatic variables had significant effects on bufogenin production (ri>0.5), while the plastic response of bufogenin toxin to various climate factors was highly variable. The most important climatic driver of total bufogenin production was precipitation: the bufogenin concentration increased with increasing precipitation. This study indicated that the evolution of phenotypic plasticity in chemical defences may depend at least partly on the geographic variation of defensive toxins and their climatic context.

Cloning, prokaryotic expression and function of the Bufo bufo gargarizans 3ß-hydroxysteroid dehydrogenase (3ßHSD) gene.

Bufadienolides, one kind of steroids, are the major active component secreted by ear-side gland of Bufo species. Preliminary studies on high-throughput transcriptome sequencing about B. bufo gargarizans showed that the expression of 3ß-Hydroxysteroid dehydrogenase (3ßHSD) in ear-side gland was nearly 20 times higher than that in liver. The enzyme 3ßHSD is an essential step in the biosynthesis of steroid such as progesterone, estrogens and androgens in steroidogenic tissues. Accordingly, 3ßHSD is probably an important enzyme involved in the biosynthesis of bufadienolides. In this study, Bbg-3ßHSD cDNA was cloned from the ear-side gland of B. bufo gargarizans. Genetic engineering techniques were used to construct a recombinant prokaryotic fusion expression plasmid pCOLD-Bbg3ßHSD which was introduced into E. coli BL21 for prokaryotic expression. Bbg-3ßHSD has an open reading frame (ORF) of 1134 bp and encodes 377 amino acid residues. The speculated protein molecular weight is 42.8 kDa and its theoretical isoelectric point is 8.68. Amino acid sequence homologous analysis showed that Bbg-3ßHSD was highly homologous to the 3ßHSD protein of other species. Phylogenetic tree showed the highest similarity between Bbg-3ßHSD and 3ßHSD from Rana rugosa. The optimized expression of recombinant Bbg-3ßHSD were achieved by inducing with 0.1 mmol L-1 IPTG at 15 °C for 20 h. Enzymatic activity in vitro shows that pregnenolone and dehydroepiandroesterone could be 3ß-oxidized by Bbg-3ßHSD when NAD+ was used as the coenzyme. Enzymatic properties showed that the optimum reaction temperature of recombinant Bbg-3ßHSD was 40 °C, the optimum pH was 8.5, and the optimum coenzyme concentration was 1.5 mmol L-1.

A strategy for the metabolomics-based screening of active constituents and quality consistency control for natural medicinal substance toad venom.

Natural medicinal substances (NMS) have great inconsistency due to chemical variability, seriously limiting their development as therapeutics. Here, we chose toad venom with anti-tumor activities as a model and developed a pipeline of metabolomics-based screening and quality consistency control (MSQCC) as one potential solution to this long-standing problem. Our study firstly exemplified the power of the co-correlation screen of metabolomic and biological profiles of 180 fractions prepared from natural heterogeneous venom samples, to identify a series of bufadinolides as quality control markers for cancer cell inhibition. The next quantitative monitoring of these markers revealed great batch-to-batch variation of toad venoms. Finally, we developed a marker-based blending program (Markers-NMBT) to normalize heterogeneity of NMS. It created the blends for the conversion of the unqualified venoms with high variation in the contents of bufadienolides, into qualified products consistent with the reference. Thus, this work provides a strategy for rapid, large-scale discovery, quantification and application of quality control markers to ensure batch-to-batch consistency, and can be a crucial technology in the development of modern NMS preparations.

Distribution of NADPH-diaphorase reactivity in the central nervous system of the common toad (Bufo bufo).

We examined the distribution of nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d)-reactive elements in the central nervous system (CNS) of the common toad, Bufo bufo. The investigation involved adult male and female toads collected during the breeding season. Labeled neurons of different morphological appearances (weakly or darkly stained, unipolar, bipolar, and multipolar) and fibers were observed across all subdivisions of the amphibian brain. Overall, a similar distribution of NADPH-d-labeled neurons was observed in the brain of male and female toads. In the secondary prosencephalon NADPH-d-labeled neurons were observed in the olfactory bulbs, pallial regions, nucleus accumbens, diagonal band of Broca, septum, striatum, amygdala, suprachiasmatic and magnocellular preoptic nuclei, dorsal and ventral hypothalamus. In the diencephalon, NADPH-d-positive neurons were seen in the anterior thalamic nuclei, ventromedial and ventrolateral nuclei, central and lateral thalamic nuclei, posterior tubercle, posterodorsal division of the lateral thalamic nucleus, and in the pretectal and pretoral gray. In the mesencephalon, heavily stained neurons were present in the anterodorsal and anteroventral tegmental nuclei, magnocellular, principal and laminar nuclei of the torus semicircularis, and nucleus profundus mesencephali. In the isthmus, stained cells were observed medially and ventrally in the posterodorsal and posteroventral tegmental nuclei. In the rhombencephalon, numerous NADPH-d-stained neurons were distributed in the cerebellar nucleus, sensory and descending trigeminal nuclei, motor nuclei of the glossopharyngeal and vagus nerves, the nucleus of the solitary tract, nuclei of the hypoglossal and octaval nerves, dorsal column nucleus, central gray region, and in reticular formation. However, the complete absence of NADPH-d-stained neurons in the cerebellar cortex was an unusual feature observed in this study. The widespread distribution of NADPH-d staining in diverse cell types, belonging to a variety of neuronal systems suggests a widespread role for NADPH-d in modulating diverse functions, including sensory coding in the amphibian nervous system.

A novel cathelicidin from Bufo bufo gargarizans Cantor showed specific activity to its habitat bacteria.

Toad Bufo bufo gargarizans Cantor is still used in China as traditional Chinese medicine. However, present investigations on its skin secretions were mainly focused on the bufadienolides, the proteins/peptides contained in the secretions are largely unknown. A cDNA encoding a novel cathelicidin termed BG-CATH was identified by analysis of the toad skin transcriptome. The BG-CATH precursor was predicted to have 2 possible cleavage sites following dibasic cleavage signals at its C-terminal, which will generate two mature peptides, BG-CATH37 and BG-CATH(5-37). Phylogenetic analysis suggests that amphibian cathelicidins might evolve from common ancestors. The two predicted mature cathelicidins from B. bufo gargarizans were synthesized and both of them showed weak antimicrobial activities against human pathogenic bacteria Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus (MIC ≥ 200 µg/mL). However, BG-CATH37 and BG-CATH(5-37) had strong antimicrobial activities against aquatic bacteria of Vibrio splendidus, Streptococcus iniae and Aeromorus hydrophila, which were common microorganisms in the habitat of B. bufo gargarizans (MIC 3.125-40 µg/mL). BG-CATH37 and BG-CATH(5-37) showed no hemolytic activity even at high concentrations (400 µg/mL). CD spectra analysis suggested that structure rigidity of BG-CATH37 and BG-CATH(5-37) might play an important role to regulate their biological activities. Selective antimicrobial activity against habitat microorganisms might reflect the adaptation of amphibians to their living environments.

Thallium in spawn, juveniles, and adult common toads (Bufo bufo) living in the vicinity of a zinc-mining complex, Poland.

A breeding population of the common toad Bufo bufo living in the vicinity of a Zn-Pb smelting works in Bukowno, Poland was studied for the presence of thallium. Tl concentration was measured in the bottom sediments of the spawning pond, in the laid eggs, in juveniles after metamorphosis, and in the selected tissues of the adult individuals. A very high concentration of Tl was detected in the spawn (13.97 ± 8.90 mg/kg d.w.). In 50% of the spawn samples, levels exceeded 20 mgTl/kg d.w. The issue of maternal transfer of thallium from females to oocytes is discussed. Due to a significant accumulation of thallium, spawn analysis can be used as a sensitive indicator of the presence of this element in the environment and may replace more invasive methods that involve the killing of adult animals. In those regions that are abundant in Zn-Pb ores, the spawn of amphibians may be a very important source of thallium contamination for predators. From among all tissues of the Bukowno adult toads, the livers have shown the highest accumulation of thallium (mean 3.98 mg/kg d.w. and maximum value--18.63). For as many as 96.5% of livers, concentrations exceeded 1.0 mgTl/kg d.w. which is treated as indicative of poisoning.

Histochemical and structural characterization of egg extra-cellular matrix in bufonid toads, Bufo bufo and Bufotes balearicus: molecular diversity versus morphological uniformity.

The extra-cellular matrix of fertilized eggs in the bufonid toads Bufo bufo and Bufotes balearicus was studied to clear the relationships between structural and molecular diversity. Histochemical (PAS, AB pH 2.5 and pH 1.0, Beta-elimination PAS) and lectin-histochemical (Con A, WGA, Succinyl-WGA, PNA, RCA-1, DBA, SBA, AAA, UEA-I, LTA) techniques were used and the observations were made under light and electron microscopy. Both species present a fertilization envelope (FE) and two jelly layers (J1 and J2). The fibers of J2 are shared among the eggs of a clutch in a jelly ribbon. The FE of both species presents neutral glycoproteins, mostly N-linked. In B. bufo there are also residuals of mannose and/or glucose and N-acetylglucosamine. In the FE fibers run parallel to egg's surface or are in bundles or looser hanks with no clear orientation. The J1 layer of both species presents sialosulfoglycoproteins, mostly O-linked, with lactosaminylated, galactosaminylated, glycosaminylated, and fucosylated residuals. A lower amount of galactosaminylated residuals is observed in B. balearicus in respect to B. bufo, whereas the opposite is seen in the amount of fucosylated residuals. The J2 layer is similar in composition to J1 but in B. balearicus there are no glucosaminylated residuals. J layers present fibers and granules that reduce towards J2 . Several microorganisms, in particular blue algae, are observed in the J2 layer of both species. In respect to other species, B. bufo and B. balearicus have a lower number of jelly layers, but a comparable number of glycan types.

Effects of spirotetramat on the acute toxicity, oxidative stress, and lipid peroxidation in Chinese toad (Bufo bufo gargarizans) tadpoles.

The aim of this work was to evaluate the potential effects of antioxidant and lipid peroxidation parameters as indicators of exposure to spirotetramat and effects of acute toxicity in the Chinese toad Bufo bufo gargarizans. The results of an acute toxicity test showed that the 72 and 96 h median lethal concentrations (LC(50)) of spirotetramat for tadpoles were 6.98 and 6.45 mg/L, respectively. It indicated that the spirotetramat was moderate toxicity to Chinese toad tadpoles. In a sub-lethal toxicity test, the superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA) contents were determined after exposure to 0.03, 0.06, 0.13, 0.65, and 3.23 mg/L for 4, 15, and 30 days. SOD activity significantly in all experimental groups except the highest concentration group increased on day 4 but decreased on days 15 compared with that of the acetone control (P < 0.05). The most sensitive parameters was GSH-Px activity, which significantly increased on day 4, but was inhibited and decreased after prolonged exposure for 15 and 30 days except the lowest concentration treatment group (P < 0.05). The MDA content significantly decreased on day 30 (P < 0.05). During the entire experimental period, sub-lethal doses spirotetramat caused oxidative stress and lipid peroxidation in B. gargarizans tadpoles. These results indicate that sub-lethal even non-lethal spirotetramat are potentially toxic to amphibians. The information presented in this study will be helpful for understanding oxidative stress induced by spirotetramat in aquatic organisms.

The supercritical CO2 extract from the skin of Bufo bufo gargarizans Cantor blocks hepatitis B virus antigen secretion in HepG2.2.15 cells.

The skin of Bufo bufo gargarizans Cantor has long been used for the treatment of hepatitis B in China and supercritical carbon dioxide extraction (SC-CO2) is widely used in extracting active ingredients from natural products. The aim of present study was to assess the anti-hepatitis B virus (HBV) effect of the supercritical CO2 extract from the skin of Bufo bufo gargarizans Cantor (SCE-BC). Cytotoxicity of SCE-BC was analyzed using an MTT [3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide] assay in HepG2.2.15 cells. The hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg), and hepatitis B core-related antigen (HBcrAg) concentrations in cell culture medium were determined by chemiluminescent enzyme immunoassay. HBV mRNA in cells was determined using real-time polymerase chain reaction. SCE-BC concentrations below 10(-2) µg/mL had no significant toxicity to HepG2.2.15 cells. SCE-BC at 10(-4) µg/mL effectively inhibited the secretion of HBeAg by 23.36% on day 6. It was more potent than the positive control lamivudine (100 µg/mL) in terms of the inhibition of HBeAg and HBcrAg secretion on day 6. Consistent with the HBV antigen reduction, HBV mRNA expression was markedly inhibited in comparison to the control when HepG2.2.15 cells were treated with SCE-BC. Moreover, SCE-BC had greater inhibitory activity with respect to HBeAg than to HBsAg. Since HBeAg promotes immune tolerance and persistent infection during HBV infection, the present results suggest that immune tolerance induced by HBeAg might be overcome by SCE-BC. Therefore, SCE-BC warrants further investigation.

Purification of bufadienolides from the skin of Bufo bufo gargarizans Cantor with positively charged C18 column.

As a kind of promising anticancer compounds, the preparation of bufadienolides is a hot study spot. However, due to the complexity of biological sample, the purification of bufadienolides from a crude sample (toad skin) is a tough work. In this paper, we reported a new way based on positively charged C18 material (XCharge C18) to quickly separate and purify bufadienolides from toad skin. By this method, the different ionic feature of the amino acid conjugated bufadienolides (AACBs) and the free form bufadienolides (AAUBs) was firstly utilized to obtain distinct separation selectivity on the XCharge C18 column. Additionally, the peak tailing problem of AACBs on conventional C18 was resolved and better resolutions were achieved on the XCharge C18, thus, two kinds of bufadienolides on one column were successfully purified respectively. Taking F13 as an example, the method was validated by liquid chromatography-mass spectrometry (LC-MS), and then 4 AACBs as well as 4 AAUBs were simultaneously purified by preparative XCharge C18. In addition, the application of this method in other fractions was also validated. The results suggested that the developed method is a practical and promising tool for efficient separation and purification of bufadienolides from toad skin.

Cd, Cu, Zn, Se, and metallothioneins in two amphibians, Necturus maculosus (Amphibia, Caudata) and Bufo bufo (Amphibia, Anura).

The accumulation of cadmium, its affinity for metallothioneins (MTs), and its relation to copper, zinc, and selenium were investigated in the experimental mudpuppy Necturus maculosus and the common toad Bufo bufo captured in nature. Specimens of N. maculosus were exposed to waterborne Cd (85 µg/L) for up to 40 days. Exposure resulted in tissue-dependent accumulation of Cd in the order kidney, gills > intestine, liver, brain > pancreas, skin, spleen, and gonads. During the 40-day exposure, concentrations increased close to 1 µg/g in kidneys and gills (0.64-0.95 and 0.52-0.76; n = 4), whereas the levels stayed below 0.5 in liver (0.14-0.29; n = 4) and other organs. Cd exposure was accompanied by an increase of Zn and Cu in kidneys and Zn in skin, while a decrease of Cu was observed in muscles and skin. Cytosol metallothioneins (MTs) were detected as Cu,Zn-thioneins in liver and Zn,Cu-thioneins in gills and kidney, with the presence of Se in all cases. After exposure, Cd binding to MTs was clearly observed in cytosol of gills as Zn,Cu,Cd-thionein and in pellet extract of kidneys as Zn,Cu,Cd-thioneins. The results indicate low Cd storage in liver with almost undetectable Cd in liver MT fractions. In field trapped Bufo bufo (spring and autumn animals), Cd levels were followed in four organs and found to be in the order kidney > liver (0.56-5.0 µg/g >0.03-0.72 µg/g; n = 11, spring and autumn animals), with no detectable Cd in muscle and skin. At the tissue level, high positive correlations between Cd, Cu, and Se were found in liver (all r > 0.80; α = 0.05, n = 5), and between Cd and Se in kidney (r = 0.76; n = 5) of autumn animals, possibly connected with the storage of excess elements in biologically inert forms. In the liver of spring animals, having higher tissue level of Cd than autumn ones, part of the Cd was identified as Cu,Zn,Cd-thioneins with traces of Se. As both species are special in having liver Cu levels higher than Zn, the observed highly preferential Cd load in kidney seems reasonable. The relatively low Cd found in liver can be attributed to its excretion through bile and its inability to displace Cu from MTs. The associations of selenium observed with Cd and/or Cu (on the tissue and cell level) point to selenium involvement in the detoxification of excessive cadmium and copper through immobilization.

[Chemical constituents from Bufonis periostracum and their antitumor activity in vitro].

Eight compounds were isolated from Bufonis periostracum by repeated column chromatography on silica gel, ODS and Sephadex LH-20 and their structures were characterized as palmitatic acid cholesteryl ester (1), cholesterol (2), 5alpha, 8alpha-epidioxycholesta-6-en-3beta-ol (3), cholest-5-en-3beta, 7beta-diol (4), cholest-7-en-3beta, 5alpha, 6beta-triol (5), 3-octaddecyloxy-1, 2-propanediol (6), isisamide (7) and bufothionine (8) on the base of spectral analysis. Compounds 1-8 were isolated from Bufonis periostracum for the first time and compounds 3, 5, 6, 7 were obtained from Bufo bufo gargarizans and Bufo genus for the first time. The bioassays showed all tested samples displayed no antitumor activity against the cell lines such as A549, BeL 7402, HGC-27 and HL-60, except the control compound bufalin.

Ammonia as a pheromone in anuran tadpoles.

In laboratory experiments, conspecific excretions and ammonia solutions evoked avoidance reactions in tadpoles of three anuran species, Bufo bufo, Rana temporaria, and Rana arvalis. A differential sensitivity of ammonia chemoreception was determined for two anuran species. For Bufo bufo tadpoles, these characteristics at ammonia background concentration of 0.2 mg/l lied in the range 150 % < dI/I < 500 %, and for background of 0.4 mg/l the value lied in the range 400 % < dI/I < 500 %. For Rana temporaria tadpoles, differential threshold against ammonia background concentration of 0.15 mg/l was close to 200 % and against background ammonia concentration of 1.1 mg/l was close to 100 %. These results suggest that such sensitivity of both anurans is sufficient for using ammonia in intra- and interspecies communication.

Antitumor activity of extracts and compounds from the skin of the toad Bufo bufo gargarizans Cantor.

The skin of the toad Bufo bufo gargarizans Cantor is known to be rich in bufadienolides, peptides and alkaloids. It has been found to be a source of some extracts and biologically active compounds with antitumor activity. Cinobufacini (Huachansu), a Chinese medicine prepared from the dried toad skin, has been widely used in clinical therapy for various cancers in China. Bufadienolides, such as bufalin, cinobufagin, resibufogenin, and telocinobufagin, are the major active compounds derived from the toad skin. They are the maker biologically active compounds of cinobufagin while the antitumor activity of cinobufagin may be due to this kind of components. Experimental research has suggested that cinobufacini and its active compounds (e.g. bufalin and cinobufagin) exhibit significant antitumor activity, including inhibition of cell proliferation, induction of cell differentiation, induction of apoptosis, disruption of the cell cycle, inhibition of cancer angiogenesis, reversal of multi-drug resistance, and regulation of the immune response. Clinical data have indicated that cinobufacini may have effective anticancer activity with low toxicity and few side effects. Data to date suggest it may also enhance quality of life for patients with cancer. Thus, this review briefly summarizes recent studies on the anticancer activity of cinobufacini and some of its active compounds from the skin of the toad Bufo bufo gargarizans Cantor. This might provide additional evidence for further study of the extracts and active compounds from the toad skin in cancer treatment.

Cinobufacini, an aqueous extract from Bufo bufo gargarizans Cantor, induces apoptosis through a mitochondria-mediated pathway in human hepatocellular carcinoma cells.

AIM OF THE STUDY: Cinobufacini (Huachansu), an aqueous extract from the skin and parotid venom glands of Bufo bufo gargarizans Cantor, is a traditional Chinese medicine widely used in clinical cancer therapy in China. The present study sought to investigate the possible signaling pathway implicated in cinobufacini-induced apoptosis in the hepatocellular carcinoma cell lines HepG(2) and Bel-7402. MATERIALS AND METHODS: The effects of cinobufacini on cell proliferation of HepG(2) and Bel-7402 cells were evaluated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assays. Cell apoptosis was detected by Hoechst 33258 staining and flow cytometry analysis. The mitochondrial membrane potential (Deltapsim) and caspase-9 and -3 activity were detected using MitoCapture reagent staining and colorimetric assays, respectively. The expression of apoptosis-related proteins and release of cytochrome c were assessed by Western blot analysis. RESULTS: Cinobufacini significantly inhibited cell proliferation of both cell lines in a dose- and time-dependent manner. Marked changes in apoptotic morphology and apoptosis rates were clearly observed after cinobufacini treatment. The protein expression of Bax increased whereas that of Bcl-2 decreased, leading to an increase in the Bax/Bcl-2 ratio. Subsequently, cinobufacini disrupted the mitochondrial membrane potential (Deltapsim) and resulted in the release of cytochrome c, activation of both caspase-9 and -3, and cleavage of poly (ADP-ribose) polymerase (PARP). CONCLUSION: The present study indicated that cinobufacini can induce apoptosis of HepG(2) and Bel-7402 cells through a mitochondria-mediated apoptosis pathway.